抗人天冬酰胺合成酶单克隆抗体的制备与鉴定

发布时间：2018-08-09 09:14

【摘要】： 天冬酰胺合成酶属于氨基转移酶家族成员之一,可以催化合成天冬酰胺(ASN),从而为蛋白质的合成提供原料。与正常的细胞相比,某些种类的恶性淋巴细胞这种酶的活性很低,不能合成足够量的天冬酰胺,需要依靠细胞外界环境中的天冬酰胺。本研究应用已构建的原核表达质粒pMS-ASNS表达MS2-ASNS融合蛋白,应用细胞融合和杂交瘤技术制备抗人天冬酰胺合成酶单克隆抗体。获得以下结果: 1. 42℃热诱导法诱导融合蛋白MS2-ASNS的表达,融合蛋白经SDS-PAGE电泳分离、电洗脱、PBS透析获得纯化的融合蛋白MS2-ASNS,分子量约为55 kDa,与理论值一致。 2.应用纯化融合蛋白免疫BALB/C小鼠,取脾细胞应用50%的PEG 3350与SP2/0细胞融合,融合效率为88%,阳性率2.3%。分别用融合蛋白MS2-ASNS和MS2-PAI通过ELISA双筛的方法4次筛选,最终筛选出3株稳定分泌特异性抗人天冬酰胺合成酶单克隆抗体的杂交瘤细胞株,分别命名为D10-9、F4-15和F4-16。3个细胞株体外传代和冻存复苏后抗体分泌稳定。测定单克隆抗体的亚类分别为IgG1、IgG2a和IgG2a。 3.用ELISA法鉴定抗体的特异性和效价。结果表明制备的抗体有很好的特异性,细胞株D10-9分泌的抗体效价为1:2×102 ,F4-15和F4-16分泌的抗体均达到1:5×105; Western-blot等方法鉴定抗体在K562和Hela细胞系中有很高亲和力;采用IHC技术检测天然ASNS在肝癌细胞系SMMC-7721、BEL-7402、HepG2和胃癌细胞系SGC-7901、SGC-823及肺癌、宫颈癌、甲状腺癌组织中均为胞浆表达。本研究制备了抗ASNS单克隆抗体,初步检测了ASNS在肿瘤细胞中的表达,为进一步研究ASNS在中线鼻/鼻型NK/T细胞淋巴瘤中的表达奠定了基础。
[Abstract]:Asparagine synthase is a member of aminotransferase family, which can catalyze the synthesis of asparagine (ASN), and provide raw materials for protein synthesis. Compared with normal cells, the enzyme activity of some kinds of malignant lymphocytes is very low, and it can not synthesize enough asparagine, which depends on the asparagine in the cell environment. In this study, the prokaryotic expression plasmid pMS-ASNS was used to express MS2-ASNS fusion protein, and the monoclonal antibody against human asparagine synthase was prepared by cell fusion and hybridoma techniques. The following results were obtained: 1. The fusion protein MS2-ASNSwas obtained by electroeluting the fusion protein by SDS-PAGE electrophoresis. The molecular weight of MS2-ASNSwas about 55 kDa, which was in agreement with the theoretical value. BALB/C mice were immunized with purified fusion protein. Spleen cells were fused with 50% PEG 3350 and SP2/0 cells. The fusion efficiency was 88 and the positive rate was 2.3%. Three hybridoma cell lines secreting monoclonal antibodies against human asparagine synthase were screened four times by ELISA double screen method with fusion protein MS2-ASNS and MS2-PAI, respectively. The antibody secretion of D10-9 F4-15 and F4-16.3 cell lines was stable after in vitro passage and cryopreservation and resuscitation. The subclasses of monoclonal antibody were IgG1, IgG2a and IgG2a.3, respectively. The specificity and titer of antibody were identified by ELISA method. The results showed that the antibody prepared by D10-9 had good specificity, and the titer of antibody secreted by cell line D10-9 was 1:2 脳 102F4-15 and F4-16, and the antibody secreted by F4-16 reached 1:5 脳 105.The antibody was identified by Western-blot method with high affinity in K562 and Hela cell lines. IHC technique was used to detect the cytoplasmic expression of natural ASNS in hepatocellular carcinoma cell line SMMC-7721 BEL-7402 HepG2, gastric cancer cell line SGC-7901hSGC-823, lung cancer, cervical cancer and thyroid carcinoma. In this study, monoclonal antibodies against ASNS were prepared, and the expression of ASNS in tumor cells was preliminarily detected, which laid a foundation for further study of the expression of ASNS in midline nasal / nasal NK/T cell lymphoma.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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